Bifidobacterium bifidum JYBB-322 for improving mitochondrial activity of cardiomyocytes, postbiotic preparation and application thereof

Abstract

The present invention relates to the technical field of probiotics, and in particular to a strain of Bifidobacterium bifidum JYBB-322 that improves mitochondrial activity in cardiomyocytes, a postbiotic preparation and its application. Bifidobacterium bifidum JYBB‑322 was deposited with the General Microbiology Center of the China Culture Collection Administration on September 20, 2023, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 28499. The Bifidobacterium bifidum JYBB‑322 provided by the present invention has antioxidant and other effects, and can reduce the level of cell apoptosis in the body and alleviate oxidative damage to mitochondria in the body. Taking the postbiotic preparation provided by the present invention can increase the activity of cardiomyocyte mitochondria without side effects, effectively avoiding the negative effects of drug use.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to a strain of Bifidobacterium bifidum JYBB-322 that improves mitochondrial activity of cardiomyocytes, a postbiotic preparation and applications thereof. Background Art

[0002] Mitochondrial activity is crucial to heart health. Through oxidative phosphorylation, mitochondria convert nutrients such as fatty acids and glucose into adenosine triphosphate (ATP). Cardiac muscle cells require a continuous and substantial energy supply to maintain rhythmic contraction and relaxation, thereby ensuring stable blood circulation. If mitochondrial activity is impaired, ATP production is insufficient, weakening myocardial contractility and impairing the heart's pumping function. Long-term effects can lead to serious heart diseases such as heart failure.

[0003] Mitochondria also play a key role in maintaining homeostasis within cardiomyocytes. They regulate intracellular calcium homeostasis, ensuring the proper function of cardiomyocyte excitation-contraction coupling. Mitochondria also play a crucial role in regulating apoptosis. Appropriate mitochondrial activity prevents excessive apoptosis in cardiomyocytes, maintaining the stability of cardiomyocyte number and function, and effectively protecting the normal structure and function of the heart.

[0004] Given the crucial role of cardiomyocyte mitochondrial activity in maintaining heart health, researchers have been exploring various effective methods to improve it. Several drugs, such as L-carnitine, have been used to improve cardiomyocyte mitochondrial activity. L-carnitine promotes lipid metabolism, transporting long-chain fatty acids into the mitochondrial matrix for oxidative breakdown, providing more energy for the cell. This, in turn, increases cardiomyocyte mitochondrial activity and promotes cardiomyocyte metabolism. However, the use of chemical drugs also has certain drawbacks. Some patients may experience adverse reactions such as gastrointestinal discomfort and allergies after taking L-carnitine, and long-term use may also increase liver and kidney stress. Coenzyme Q10 is also commonly used to enhance mitochondrial function. It plays a vital role in cellular respiration and metabolism, helping mitochondria produce energy more efficiently. However, the effectiveness of Coenzyme Q10 supplementation varies greatly from person to person, and long-term, high-dose use may inhibit the body's ability to synthesize Coenzyme Q10.

[0005] Aerobic exercise is a natural and effective way to boost mitochondrial activity. Exercise can increase the activity of enzymes involved in energy metabolism in mitochondria, improve the efficiency of mitochondrial oxidative phosphorylation, and enhance the energy supply capacity of cardiomyocytes. However, the effects of exercise are not achieved overnight; they require long-term persistence and maintaining an appropriate intensity and frequency. Furthermore, excessive exercise may increase oxidative stress and damage mitochondria. For some people with severe heart disease or those in poor physical condition, implementing an exercise program still has certain limitations and risks.

[0006] Other studies have shown that certain strains of probiotics, such as Lactobacillus plantarum ( Lactobacillus plantarum TCI999 has demonstrated the ability to enhance mitochondrial activity in cell-based experiments (Chinese invention patent CN110734869 B). However, the effects of different probiotic strains may vary, and currently, there is a lack of Bifidobacterium bifidum that can improve mitochondrial activity in cardiomyocytes. Summary of the Invention

[0007] In response to the technical problem of the lack of Bifidobacterium bifidum capable of improving the mitochondrial activity of cardiomyocytes, the present invention provides a strain of Bifidobacterium bifidum JYBB-322 that improves the mitochondrial activity of cardiomyocytes, a postbiotic preparation and applications thereof.

[0008] The technical solutions of the present invention are as follows:

[0009] In the first aspect, the present invention provides a strain of Bifidobacterium bifidum JYBB-322 that improves mitochondrial activity in cardiomyocytes. Bifidobacterium bifidum ) JYBB-322 was deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 20, 2023, and the deposit number is CGMCC No. 28499. The 16S rDNA sequence of Bifidobacterium bifidum JYBB-322 is shown in SEQ ID No: 1.

[0010] In a second aspect, the present invention provides a postbiotic preparation for improving mitochondrial activity in cardiomyocytes, wherein the postbiotic preparation comprises the above-mentioned Bifidobacterium bifidum JYBB-322.

[0011] It should be further explained that the postbiotic preparation for improving the mitochondrial activity of cardiomyocytes provided by the present invention also contains auxiliary substances permitted in the pharmaceutical field, such as carriers, fillers, diluents, adhesives, lubricants, disintegrants, solubilizers, stabilizers, surfactants, etc.; preferably, the postbiotic preparation for improving the mitochondrial activity of cardiomyocytes provided by the present invention also contains maltodextrin.

[0012] It should be further explained that the dosage form of the postbiotic preparation for improving the mitochondrial activity of cardiomyocytes provided by the present invention can be selected from one of tablets, powders, granules, capsules, pills, sustained-release preparations, suspensions, oral liquid preparations, injections, etc., and the administration method can be oral or parenteral (intravenous injection, subcutaneous injection, etc.); preferably, the dosage form of the postbiotic preparation for improving the mitochondrial activity of cardiomyocytes provided by the present invention is a powder, and is used in conjunction with an oral administration method.

[0013] It should be further explained that the number of Bifidobacterium bifidum JYBB-322 in the postbiotic preparation for improving mitochondrial activity of cardiomyocytes provided by the present invention is 1.0×10 7 cfu / g~1.0×10 8 cfu / g; preferably, the number of Bifidobacterium bifidum JYBB-322 in the postbiotic preparation for improving mitochondrial activity of cardiomyocytes provided by the present invention is 1.0×10 8 cfu / g.

[0014] It should be further explained that the postbiotic preparation for improving mitochondrial activity of cardiomyocytes provided by the present invention is prepared according to the following preparation method:

[0015] The bacterial suspension of Bifidobacterium bifidum JYBB-322 was heat-inactivated, concentrated, and freeze-dried to obtain postbiotic powder. The postbiotic powder was mixed with maltodextrin to prepare a mixture containing 1.0×10 7 cfu / g~1.0×10 8 cfu / g of postbiotic preparations.

[0016] It should be further explained that the bacterial solution of Bifidobacterium bifidum JYBB-322 was prepared according to the following preparation method:

[0017] The activated single colony of Bifidobacterium bifidum JYBB-322 was transferred into MRS liquid culture medium and cultured at a constant temperature of 37°C to prepare seed liquid;

[0018] The seed liquid was inoculated into MRS liquid culture medium at an inoculum rate of 1% by mass, and cultured at a constant temperature of 37°C to obtain a bacterial liquid.

[0019] It should be further explained that the MRS liquid culture medium was prepared according to the following preparation method:

[0020] Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, and 0.2 g of magnesium sulfate heptahydrate, dissolve them in distilled water and make up to 1 L. Adjust the pH of the solution to 6.8, and sterilize at 121°C and 0.1 MPa for 20 min.

[0021] In a third aspect, the present invention provides a use of the above-mentioned Bifidobacterium bifidum JYBB-322 in the preparation of a postbiotic preparation for improving mitochondrial activity of cardiomyocytes.

[0022] The beneficial effects of the present invention are:

[0023] The Bifidobacterium bifidum JYBB-322 provided by the present invention has antioxidant effects and can reduce the level of cell apoptosis in the body and alleviate mitochondrial oxidative damage in the body. Taking the postbiotic preparation provided by the present invention can improve the activity of cardiomyocyte mitochondria without side effects, effectively avoiding the negative effects caused by the use of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 3 is a bar graph of mitochondrial activity of cardiomyocytes of each group of mice in Example 3.

[0026] Figure 2 3 is a bar graph of the ATP content in the myocardial cells of each group of mice in Example 3.

[0027] Figure 3 4 is a bar graph showing the integrity of mitochondrial DNA in cardiomyocytes of mice in each group.

[0028] Figure 4 3 is a bar graph of the reactive oxygen species levels in the cardiomyocytes of each group of mice in Example 5.

[0029] Figure 5 3 is a bar graph showing the expression levels of Bcl-2 protein in the cardiomyocytes of each group of mice in Example 6.

[0030] Figure 6 3 is a bar graph showing the expression levels of P53 protein in the cardiomyocytes of each group of mice in Example 6.

[0031] Figure 73 is a bar graph of the expression levels of Caspase-3 protein in the cardiomyocytes of each group of mice in Example 6.

[0032] Figure 8 3 is a bar graph of the expression levels of Caspase-9 protein in the cardiomyocytes of each group of mice in Example 6.

[0033] In the figures, different lowercase letters indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0035] Example 1 Isolation and Identification of Bifidobacterium bifidum JYBB-322

[0036] 1. Strain collection and purification

[0037] (1) Sampling

[0038] In February 2021, feces of infants and young children in Zhangqiu District, Jinan City, Shandong Province were collected and transported back to the laboratory via cold chain for standby use.

[0039] (2) Isolation and purification of alternative strains

[0040] ① Take 1 g of infant feces and add it to a conical flask containing sterile saline. Stir and shake at 4°C for 30 minutes and set aside.

[0041] ② Use sterile saline to dilute the solution in step ① step by step, from 10 -1 to 10 -7 A total of 7 dilution gradients were prepared for future use.

[0042] ③ Dissolve 10 g peptone, 5 g beef extract powder, 5 g sodium acetate trihydrate, 2 g potassium dihydrogen phosphate heptahydrate, 1 mL Tween-80, 0.05 g manganese sulfate tetrahydrate, 2 g triammonium citrate, 20 g glucose, 0.2 g magnesium sulfate heptahydrate, 5 g calcium carbonate, and 15 g agar in distilled water to make up to 1 L. Adjust the pH of the solution to 6.8. Heat and mix thoroughly, then sterilize at 121°C, 0.1 MPa for 20 min. Pour the sterilized culture medium into a plate and let it cool to obtain MRS plate medium.

[0043] ④ Use a spreading rod to spread the solutions of different dilution concentrations prepared in step ② onto MRS plate culture medium, place them in an anaerobic incubator, and culture at a constant temperature of 37°C for 48 h.

[0044] ⑤ Select 22 single colonies based on the characteristics of colony diameter of 1 mm to 2 mm, round colony, neat edge, slightly white, bulge in the middle, and large calcium dissolution circle. Inoculate them onto MRS plate culture medium by streak method. Incubate at 37℃ under anaerobic conditions for 48 h. Repeat the above operation 2 to 3 times, pick single colonies, place them in glycerol tubes and store them at -70℃ as alternative strains.

[0045] (3) Screening of strains

[0046] Mitochondrial dysfunction in cardiomyocytes produces a large amount of reactive oxygen free radicals (ROS), which damage the heart. By scavenging the oxidative free radicals, we can preliminarily screen strains that have the potential to alleviate mitochondrial dysfunction.

[0047] The specific screening methods are as follows:

[0048] ① Dissolve 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of potassium dihydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, and 0.2 g of magnesium sulfate heptahydrate in distilled water and adjust the volume to 1 L. Adjust the pH of the solution to 6.8. Heat and mix thoroughly. Sterilize at 121°C, 0.1 MPa for 20 min. Let cool to obtain MRS liquid medium.

[0049] ② Pick a single colony of each of the 22 candidate strains mentioned above in an inoculation loop and inoculate them into MRS liquid culture medium respectively. Incubate at 37°C for 24 h to obtain fermentation broth for use.

[0050] ③ Transfer 400 μL of the fermentation broth prepared from 22 different strains into culture tubes. Add 1 mL of a 0.2 mM DPPH-ethanol solution to each culture tube. Incubate in the dark at room temperature for 60 minutes. Measure the absorbance (A) of the solution at 517 nm using a visible light spectrophotometer.

[0051] Transfer 400 μL of distilled water to a culture tube. Add 1 mL of a 0.2 mM DPPH-ethanol solution to the tube and incubate in the dark at room temperature for 60 minutes. Measure the absorbance (A0) of the solution at 517 nm using a visible light spectrophotometer.

[0052] The scavenging rate of active oxygen by the alternative strains was calculated according to the following formula.

[0053] Clearance rate (%) = ((A0-A) / A0) × 100%.

[0054] The clearance rates of the candidate strains were compared, and a candidate strain with the highest clearance rate for reactive oxygen species was screened out. The specific clearance rate data for reactive oxygen species of this candidate strain was 88%. The strain was identified, preserved, and used in subsequent experiments.

[0055] (4) Identification of strains

[0056] The candidate strain with the highest active oxygen scavenging rate obtained from the above screening was sent to Sangon Biotech (Shanghai) Co., Ltd. for identification.

[0057] After identification, the 16S rDNA sequence of the candidate strain (SEQ ID No: 1) is as follows:

[0058]

[0059] The identification results were compared with the database by BLAST, confirming that the candidate strain was Bifidobacterium bifidum ( Bifidobacterium bifidum Based on this, the candidate strain was named Bifidobacterium bifidum JYBB-322.

[0060] (5) Preservation of strains

[0061] Bifidobacterium bifidum JYBB-322 was deposited in the General Microbiology Center of China Culture Collection Administration. The deposit information is as follows:

[0062] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0063] Deposit date: September 20, 2023;

[0064] Deposit number: CGMCC No.28499;

[0065] Classification name: Bifidobacterium bifidum Bifidobacterium bifidum .

[0066] Example 2 Preparation of postbiotic preparations

[0067] S1: Activate Bifidobacterium bifidum JYBB-322 stored at -80°C on MRS plate culture medium. The preparation method of MRS plate culture medium is as follows: take 10 g peptone, 5 g beef extract powder, 5 g sodium acetate trihydrate, 2 g dipotassium hydrogen phosphate heptahydrate, 1 mL Tween-80, 0.05 g manganese sulfate tetrahydrate, 2 g triammonium citrate, 20 g glucose, 0.2 g magnesium sulfate heptahydrate, 5 g calcium carbonate, and 15 g agar, dissolve them in distilled water and make up to 1 L, adjust the pH value of the solution to 6.8, heat and mix, and sterilize at 121°C and 0.1 MPa for 20 min. Pour the sterilized culture medium into a plate and let it cool.

[0068] S2: Then, pick a single colony after activation and transfer it to 100 mL of MRS liquid culture medium, and culture it at a constant temperature of 37°C for 12 h to prepare the seed solution. The MRS liquid culture medium was prepared according to the following preparation method: take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, and 0.2 g of magnesium sulfate heptahydrate, dissolve them in distilled water and make the volume to 1 L, adjust the pH value of the solution to 6.8, heat and mix, sterilize at 121°C and 0.1 MPa for 20 min, and cool to obtain the result.

[0069] S3: The seed liquid was inoculated into MRS liquid culture medium (prepared in the same manner as step S2) at an inoculum rate of 1% by mass, and then cultured at a constant temperature of 37°C under anaerobic conditions for 24 h to obtain a bacterial solution.

[0070] S4: The bacterial solution was heat-inactivated at 115°C for 30 min, and then concentrated and freeze-dried to obtain the postbiotic powder;

[0071] S5: Postbiotic powder was mixed with maltodextrin (purchased from Baolingbao Biotechnology Co., Ltd.) to prepare 1.0×10 7 cfu / g, 5.0×10 7 cfu / g and 1.0×10 8 cfu / g of postbiotic preparations.

[0072] Example 3 Effects of Bifidobacterium bifidum JYBB-322 on mitochondrial activity and adenosine triphosphate content in cardiomyocytes

[0073] Ten male 6-month-old SPF Kunming mice and 40 male 6-month-old senescence-accelerated mouse prone 8 (SAMP8 mice) model mice were purchased.

[0074] After 2 weeks of adaptive feeding, 10 SPF Kunming mice were used as a control group and were treated with 100 μL of gelatin aqueous solution by gavage;

[0075] SAMP8 mice were randomly divided into 4 groups as follows:

[0076] Model group: 10 SAMP8 mice were treated with 100 μL gelatin aqueous solution by gavage only;

[0077] Group A: 10 SAMP8 mice, each day 0.02 g of 1.0×10 8 cfu / g of postbiotic preparations + 100 μL gelatin aqueous solution by gavage;

[0078] Group B: 10 SAMP8 mice, each day 0.04 g of 1.0×10 8 cfu / g of postbiotic preparations + 100 μL gelatin aqueous solution by gavage;

[0079] Group C: 10 SAMP8 mice, each day 0.06 g of 1.0×10 8 cfu / g of postbiotic preparations + 100 μL gelatin aqueous solution by gavage;

[0080] The preparation method of the gelatin aqueous solution is as follows: add 0.1 g of gelatin to 1000 mL of pure water, and then heat the solution in a water bath at 60°C to completely dissolve the gelatin.

[0081] The drinking water of each group of mice was changed every two days, and they continued to be fed maintenance feed during the treatment period, which lasted for 2 weeks.

[0082] After 2 weeks of feeding, mice in each group were dissected and fresh heart tissue was obtained. Mitochondria were extracted according to the operating procedures of the mitochondrial extraction kit (Solebo). The heart tissue was immunofluorescently stained using a fluorescent probe staining method (JC-1). The mitochondrial membrane potential of cardiomyocytes was then detected by flow cytometry, and mitochondrial activity was calculated using FlowJo analysis software.

[0083] The results are as follows Figure 1 As shown, the mitochondrial activity in the model group was significantly lower than that in the control group, indicating that mitochondrial activity was impaired in the aging-accelerated mouse model. Groups A, B, and C were all gavaged with a postbiotic preparation containing Bifidobacterium bifidum JYBB-322. Group A, which used a lower dose (0.02 g) of the postbiotic preparation, had higher mitochondrial activity than the model group, preliminarily demonstrating the postbiotic preparation's effect on enhancing mitochondrial activity. The mitochondrial activity in Groups B (0.04 g postbiotic preparation) and C (0.06 g postbiotic preparation) was significantly higher than that in the model group and closer to the level of the control group, strongly demonstrating that the postbiotic preparation prepared in Example 2 can effectively enhance mitochondrial activity in cardiomyocytes of aging-accelerated mice.

[0084] Fresh heart tissues from each group were ground and then the adenosine triphosphate content in the cardiomyocytes of each group was determined by colorimetry according to the instructions of the ATP content determination kit (Nanjing Jiancheng).

[0085] The results are as follows Figure 2 As shown, it can be seen that the ATP content in the model group was significantly lower than that in the control group, indicating that the rapid aging mouse model has insufficient ATP production. Group A was gavaged with a lower dose (0.02 g) of the postbiotic preparation, and its ATP content was higher than that of the model group, indicating that the postbiotic preparation has a certain effect on increasing the ATP content. Groups B and C were gavaged with 0.04 g and 0.06 g of the postbiotic preparation, respectively, and the ATP content of both groups was significantly higher than that of the model group, and was relatively close to that of the control group. This fully proves that the postbiotic preparation prepared in Example 2 can effectively increase the ATP content of cardiomyocytes in rapidly aging mice.

[0086] Overall, Bifidobacterium bifidum JYBB-322 effectively enhanced mitochondrial activity in the cardiomyocytes of rapidly aging mice by reducing mitochondrial oxidative damage and other mechanisms, which has positive implications for maintaining energy supply and internal environmental homeostasis for heart health. Furthermore, Bifidobacterium bifidum JYBB-322 also effectively improved adenosine triphosphate production in the cardiomyocytes of rapidly aging mice, playing a positive role in maintaining cardiomyocyte energy supply.

[0087] Example 4 Effect of Bifidobacterium bifidum JYBB-322 on the integrity of mitochondrial DNA in cardiomyocytes

[0088] Mitochondrial DNA is particularly susceptible to oxidative damage due to its proximity to the electron transport chain, which produces reactive oxygen species as a byproduct. When mitochondrial DNA is damaged, fragments are released into the cytoplasm, triggering immune responses and contributing to the development of various diseases. 8-Hydroxydeoxyguanosine (8-OHdG) is a biomarker of oxidative stress, and its measurement is commonly used as an indicator of mitochondrial DNA (mtDNA) damage.

[0089] Therefore, fluorescent staining analysis of 8-hydroxydeoxyguanosine in the cardiomyocytes of each group of mice in Example 3 was performed to detect the integrity of the cardiomyocyte mitochondrial DNA. The specific steps are as follows:

[0090] (1) Sample preparation: Centrifuge the sample to be tested and transfer the precipitate to a new centrifuge tube. Wash the precipitate once with PBS, and then resuspend the cell pellet in an appropriate buffer.

[0091] (2) Prepare slides: Soak the slides in 95% alcohol for 10 min, then wash them three times with sterile deionized water for 10 min each. Allow the slides to dry, then apply a layer of poly-L-lysine to the slides.

[0092] (3) Fixation of cardiomyocytes: Drop the suspended cardiomyocytes onto a poly-L-lysine-coated glass slide and let it stand for 20 minutes to allow the cardiomyocytes to adhere to the glass slide. Then, fix the cells with methanol at room temperature and wash them three times with PBS after 10 minutes.

[0093] (4) Permeabilization: Use 3% Triton X-100 permeabilization agent (PBST) to rupture the cell membrane for 1 h to make the cell membrane permeable and facilitate the entry of antibodies into the cells.

[0094] (5) Blocking: Soak the slides in 10% goat serum for 30 min to block the binding of nonspecific antibodies to cardiomyocytes.

[0095] (6) Add primary antibody: Add 8-OHdG antibody to bind to the myocardial cell immunogen to be detected. Place the slide in a box with high humidity and place it in a refrigerator at 4°C for overnight incubation.

[0096] (7) Washing: Wash the slides with PBS buffer to remove unbound antibodies.

[0097] (8) Adding secondary antibody: Add fluorescently labeled secondary antibody to bind to the primary antibody to form a complex.

[0098] (9) Washing: Wash the slides with PBS buffer to remove unbound secondary antibodies.

[0099] (10) Nuclear staining: Add DAPI nuclear stain, stain the cell nucleus for 5 min, and wash away the floating color with PBS buffer.

[0100] (11) Sealing: Use anti-fluorescence attenuation mounting medium to seal the slides.

[0101] (12) Microscope observation: Observe the slides under a fluorescence microscope to detect the immunofluorescence signal in the sample. Calculate the mitochondrial DNA damage rate according to the formula: damage rate = number of cells that stimulate red fluorescence in the image / total number of cells in the image × 100%.

[0102] The results are as follows Figure 3 As shown, the mitochondrial DNA damage rate in the control group was relatively low, indicating that the mitochondrial DNA in the cardiomyocytes of normal mice was less oxidatively damaged. The mitochondrial DNA damage rate in the model group was significantly higher than that in the control group, indicating that the mitochondrial DNA in the aging-accelerated mouse model had suffered more severe oxidative damage. This is consistent with the fact that mitochondria are close to the electron transport chain and are susceptible to attack by reactive oxygen species. Group A, which received 0.02 g of the postbiotic preparation by gavage, had a lower mitochondrial DNA damage rate than the model group, demonstrating that the postbiotic preparation has a certain effect in reducing mitochondrial DNA damage. Groups B (0.04 g of the postbiotic preparation) and C (0.06 g of the postbiotic preparation) had even lower mitochondrial DNA damage rates, with Group B having the lowest damage rate. This fully demonstrates that the Bifidobacterium bifidum JYBB-322 provided by the present invention can significantly reduce oxidative damage to the mitochondrial DNA in the cardiomyocytes of aging-accelerated mice through its antioxidant activity, which has positive implications for maintaining mitochondrial DNA integrity and heart health. The postbiotic preparation prepared in Example 2 can effectively reduce the mitochondrial DNA damage rate in the cardiomyocytes of aging-accelerated mice.

[0103] Example 5 Effect of Bifidobacterium bifidum JYBB-322 on Reactive Oxygen Species Levels in Cardiomyocytes

[0104] The mice in each group in Example 3 were used as experimental subjects. Fresh heart tissues were obtained by dissection, and the reactive oxygen species levels in the myocardial tissues of each group of mice were detected according to the instructions of the ROS detection kit (Nanjing Jiancheng).

[0105] The results are as follows Figure 4 As shown, it can be seen that the reactive oxygen level in the control group is relatively low, indicating that the oxidative stress in the cardiomyocytes of normal mice is at a low level. The reactive oxygen level in the model group was significantly higher than that in the control group, indicating that there is a significant increase in oxidative stress in the rapid aging mouse model, which can cause damage to cell structures such as mitochondria. Group A was gavaged with 0.02 g of the postbiotic preparation, and its reactive oxygen level was lower than that of the model group, showing that the postbiotic preparation has a certain effect in reducing the reactive oxygen level in cardiomyocytes. The reactive oxygen levels of Group B (0.04 g postbiotic preparation) and Group C (0.06 g postbiotic preparation) were further reduced, and the reactive oxygen level in Group C was relatively lower. This fully proves that the postbiotic preparation prepared in Example 2 can effectively reduce the reactive oxygen level in the cardiomyocytes of rapidly aging mice.

[0106] In summary, Bifidobacterium bifidum JYBB-322 significantly reduced the level of reactive oxygen species in the cardiomyocytes of rapidly aging mice by exerting its antioxidant effect, alleviated the damage of oxidative stress to cell structures such as mitochondria, and has positive significance for maintaining the homeostasis of the internal environment of cardiomyocytes and heart health.

[0107] Example 6 Effects of Bifidobacterium bifidum JYBB-322 on the Expression Levels of Apoptosis-Related Proteins (Bcl-2, P53, Caspase-3, and Caspase-9) in Cardiomyocytes

[0108] The mice in each group described in Example 3 were used as experimental subjects. Fresh heart tissues were obtained by dissection and protein was extracted using a BCA protein extraction kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.). The expression of Bcl-2, P53, Caspase-3, and Caspase-9 proteins in mouse cardiomyocytes was then detected using the Western Blot method.

[0109] The specific steps are as follows:

[0110] S1. Sample preparation: Prepare protein samples by lysing cells or tissues and extracting proteins.

[0111] S2. Protein quantification: Use the BCA protein extraction kit to determine the protein concentration of the sample.

[0112] S3. Protein separation: SDS-PAGE gel preparation kit (Shanghai Biotech Co., Ltd.) was used to prepare gels, and proteins were separated by electrophoresis.

[0113] S4. Protein transfer: Transfer the separated proteins from the gel to the PVDF membrane using a semi-dry transfer system.

[0114] S5. Blocking: Incubate nonspecific binding sites on the membrane in a blocking solution containing 5% bovine serum albumin.

[0115] S6. Primary antibody incubation: Incubate with the specific primary antibody for the detection protein.

[0116] S7. Secondary antibody incubation: Add secondary antibody labeled with horseradish peroxidase for incubation, and the secondary antibody will bind to the primary antibody.

[0117] S8. Signal detection: The signals generated by the bound secondary antibody were detected, and then the signals were visualized and analyzed using a gel imaging system (Thermo Fisher Scientific).

[0118] S9. Data analysis: Grayscale analysis of the signal emitted by the target protein band was performed in the image analysis software Image J to quantify the signal intensity of the target protein.

[0119] The expression levels of Bcl-2 protein in the cardiomyocytes of mice in each group were as follows Figure 5 As shown, the control group showed higher levels of Bcl-2 protein expression. Bcl-2 is an anti-apoptotic protein, indicating that normal mouse cardiomyocytes have a strong anti-apoptotic capacity. The model group showed significantly lower Bcl-2 protein expression than the control group, indicating that the cardiomyocytes of the aging-accelerated mouse model have decreased anti-apoptotic capacity and are more susceptible to apoptosis. Group A, administered 0.02 g of the postbiotic preparation by gavage, showed higher Bcl-2 protein expression levels than the model group, indicating that the postbiotic preparation can, to a certain extent, increase Bcl-2 protein expression in cardiomyocytes and enhance anti-apoptotic capacity. Bcl-2 protein expression levels were further elevated in Groups B (0.04 g of the postbiotic preparation) and C (0.06 g of the postbiotic preparation), with the Bcl-2 protein expression level in Group C being close to that of the control group. This fully demonstrates that the postbiotic preparation prepared in Example 2 can effectively increase Bcl-2 protein expression levels in cardiomyocytes of aging-accelerated mice.

[0120] The expression levels of P53 protein in the cardiomyocytes of mice in each group were as follows Figure 6As shown, it can be seen that the expression level of P53 protein in the model group was significantly higher than that in the control group. P53 protein is an important tumor suppressor protein and also plays a key role in the regulation of cell apoptosis. The high expression in the model group reflects that the cardiomyocytes of rapidly aging mice may have an abnormal apoptosis regulation state. Group A was gavaged with 0.02 g of the postbiotic preparation, and its P53 protein expression level was lower than that of the model group, indicating that the postbiotic preparation can preliminarily regulate the expression of P53 protein. The expression levels of P53 protein in Group B (0.04 g postbiotic preparation) and Group C (0.06 g postbiotic preparation) were further reduced and were close to the expression levels of the control group, fully demonstrating that the postbiotic preparation prepared in Example 2 can effectively reduce the expression level of P53 protein in the cardiomyocytes of rapidly aging mice.

[0121] The expression levels of Caspase-3 protein in the cardiomyocytes of mice in each group were as follows Figure 7 As shown, the model group showed significantly higher Caspase-3 protein expression levels than the control group. Caspase-3 is a key executioner of apoptosis, and its high expression indicates a higher level of cardiomyocyte apoptosis in the aging-accelerated mouse model. Group A, administered 0.02 g of the postbiotic preparation by gavage, showed lower Caspase-3 protein expression levels than the model group, indicating that the postbiotic preparation can initially inhibit Caspase-3 protein expression in cardiomyocytes, thereby reducing apoptosis. Caspase-3 protein expression levels in Groups B (0.04 g of the postbiotic preparation) and C (0.06 g of the postbiotic preparation) were further reduced and closer to those in the control group, fully demonstrating that the postbiotic preparation prepared in Example 2 can effectively inhibit Caspase-3 protein expression in cardiomyocytes of aging-accelerated mice.

[0122] The expression levels of Caspase-9 protein in the cardiomyocytes of mice in each group were as follows Figure 8 As shown, the model group showed significantly higher Caspase-9 protein expression levels than the control group. Caspase-9 is a key initiating protein in the intrinsic apoptosis pathway, and its high expression indicates that the intrinsic apoptosis pathway in cardiomyocytes of the aging-accelerated mouse model is strongly activated. Group A, which received 0.02 g of the postbiotic preparation via gavage, showed lower Caspase-9 protein expression levels than the model group, indicating that the postbiotic preparation can inhibit Caspase-9 protein expression in cardiomyocytes to a certain extent, thereby inhibiting the activation of the intrinsic apoptosis pathway. Caspase-9 protein expression levels in Groups B (0.04 g of the postbiotic preparation) and C (0.06 g of the postbiotic preparation) were further reduced and closer to those in the control group, fully demonstrating that the postbiotic preparation prepared in Example 2 can effectively inhibit Caspase-9 protein expression in cardiomyocytes of aging-accelerated mice.

[0123] In summary, it can be seen that after the intervention of Bifidobacterium bifidum JYBB-322, the expression level of Bcl-2 protein in mouse cardiomyocytes increased, the expression level of P53 protein in mouse cardiomyocytes decreased, the expression of Caspase-3 protein and Caspase-9 protein was inhibited, and the anti-apoptosis ability of rapidly aging mouse cardiomyocytes was enhanced, indicating that Bifidobacterium bifidum JYBB-322 can improve the mitochondrial activity of cardiomyocytes, avoid mitochondrial damage, and effectively prevent cell apoptosis caused by insufficient adenosine triphosphate production, thereby achieving the positive effects of protecting heart health, maintaining stable heart function, and delaying heart aging.

[0124] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A strain of Bifidobacterium bifidum JYBB-322 for improving mitochondrial activity in cardiomyocytes, characterized in that: Bifidobacterium bifidum ( Bifidobacterium bifidum ) JYBB-322 is deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is September 20, 2023, and the deposit number is CGMCC No. 28499.

2. A postbiotic preparation for improving mitochondrial activity in cardiomyocytes, characterized in that: The postbiotic preparation comprises the Bifidobacterium bifidum JYBB-322 according to claim 1, and the postbiotic preparation is prepared according to the following preparation method: The bacterial solution of Bifidobacterium bifidum JYBB-322 was heat-inactivated, concentrated, and freeze-dried to obtain postbiotic powder, which was then mixed with maltodextrin to prepare a solution containing 1.0×10 7 cfu / g~1.0×10 8 cfu / g of postbiotic preparations.

3. A postbiotic preparation for improving mitochondrial activity in cardiomyocytes according to claim 2, characterized in that: The dosage form of postbiotic preparations is powder.

4. A postbiotic preparation for improving mitochondrial activity in cardiomyocytes according to claim 2, characterized in that: The number of Bifidobacterium bifidum JYBB-322 cells in the postbiotic preparation was 1.0×10 8 cfu / g.

5. A postbiotic preparation for improving mitochondrial activity in cardiomyocytes according to claim 2, characterized in that: The bacterial solution of Bifidobacterium bifidum JYBB-322 was prepared according to the following preparation method: The activated single colony of Bifidobacterium bifidum JYBB-322 was transferred into MRS liquid culture medium and cultured at a constant temperature of 37°C to prepare seed liquid; The seed liquid was inoculated into MRS liquid culture medium at an inoculum rate of 1% by mass, and cultured at a constant temperature of 37°C to obtain a bacterial liquid.

6. A postbiotic preparation for improving mitochondrial activity in cardiomyocytes according to claim 5, characterized in that: MRS liquid culture medium was prepared according to the following preparation method: Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, and 0.2 g of magnesium sulfate heptahydrate, dissolve them in distilled water and make up to 1 L. Adjust the pH of the solution to 6.8, and sterilize at 121°C and 0.1 MPa for 20 min.

7. Use of the Bifidobacterium bifidum JYBB-322 according to claim 1 in the preparation of a postbiotic preparation for improving mitochondrial activity in cardiomyocytes.

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